1. Field of the Invention
The present invention relates generally to techniques for detecting and measuring levels of chemical compounds found in biological tissue. More specifically, the invention relates to a method and apparatus for Raman imaging of carotenoids and related chemical substances in biological tissue, such as macular carotenoid pigments, which can be used as a diagnostic aid for assessing the risk of disease.
2. Background Technology
Over the past few years, there has been increasing interest in studying the role of macular carotenoid pigments in the human retina. The macular pigments are comprised of the carotenoid species lutein and zeaxanthin, which are concentrated in the macula lutea. The macula lutea includes about a 5 mm diameter region of the retina essential for highest visual acuity and color vision. The macular pigments may be of fundamental importance in the treatment and prevention of age-related macular degeneration (AMD), a leading cause of blindness in the elderly. Absorbing in the blue-green spectral range, these pigments are thought to act as filters attenuating photochemical damage and/or image degradation caused by the phototoxic effect of blue/green light components reaching the retina. In addition, it is speculated that these pigments may play a protective role as free radical scavenging antioxidants.
To facilitate AMD research, a noninvasive, reliable and objective detection method for the macular pigments in the living human macula is needed. A noninvasive method for the measurement of carotenoid levels in the macular tissue of the eye is described in U.S. Pat. No. 5,873,831, the disclosure of which is herein incorporated by reference. In this method, levels of carotenoids and related substances are measured by a technique known as resonance Raman spectroscopy. This is a technique which can identify the presence and concentration (provided proper calibration is performed) of certain chemical compounds. In this technique, nearly monochromatic light is incident upon the sample to be measured, and the inelastically scattered light which is of a different frequency than the incident light is detected and measured. The frequency shift between the incident and scattered light is known as the Raman shift, and the shift corresponds to an energy which is the “fingerprint” of the vibrational or rotational energy state of certain molecules. Typically, a molecule exhibits several characteristic Raman active vibrational or rotational energy states, and the measurement of the molecule's Raman spectrum thus provides a fingerprint of the molecule, i.e., it provides a molecule-specific series of spectrally sharp vibration or rotation peaks. The intensity of the Raman scattered light corresponds directly to the concentration of the molecule(s) of interest. The resonance Raman technique described in U.S. Pat. No. 5,873,831 can be used to measure the levels of the carotenoids lutein and zeaxanthin, two chemicals which are associated with healthy macular tissue of the human eye.
Currently the most commonly used non-invasive method for measuring macular pigment levels is a psychophysical heterochromatic flicker photometric test involving color intensity matching of a light beam aimed at the fovea and another aimed at the perifoveal area. However, this method is subjective, time intensive, and requires an alert, cooperative subject with good visual acuity. Its repeatability depends on the understanding by the subject of the task involved. Thus, the usefulness of this method for assessing macular pigment levels in the elderly population most at risk for AMD and in subjects with macular pathologies is severely limited.
In another approach, an objective detection method for macular pigments is employed based on spectral fundus reflectance. This method has the advantage that it can be used both for the normal and abnormal human retina. In one variation of this method, the reflectance across the visual spectrum is measured quasi-simultaneously and estimates for macular pigment concentrations are obtained from a fit of the measured spectra with calculated spectra derived from a detailed optical model for foveal reflection and absorption. In imaging variations of this technique, an imaging fundus reflectometer or a modified scanning laser ophthalmoscope (SLO) is used to generate reflectance maps for two different scanning laser wavelengths, and the macular pigment distributions and concentrations are derived by digital image subtraction. The SLO measurements of macular pigments have been shown to provide more reliable results than spectral fundus reflectance or psychophysical measurements. However, the spectral fundus reflectance and SLO methodologies suffer from a lack of chemical specificity in their measurements due to the presence of multiple broadly absorbing substances in the retina.